The long term interest of my laboratory is to investigate positive- stranded viruses with respect to the regulation of pathogenesis and reversion of non-pathogenic strains of viruses to pathogenicity. The education of undergraduate and graduate students is a high priority. At present, two viruses are being studied in my laboratory, mengovirus and transmissible gastroenteritis virus. Mengovirus is a member of the Cardiovirus group of the Picornaviridae along with encephalomyocarditis (EMC), Columbia SK, Theiler's encephalitis and Maus Elberfeld viruses. The heat stable strain of mengovirus (37A) in this project agglutinates type O human red blood cells, and causes encephalomyelitis and hind limb paralysis in mice. We have prepared and partially characterized a library of 24 temperature-sensitive (ts) mutants derived by mutagenesis with nitrosoguanidine and 11 ts mutants by acriflavine mutagenesis. The objective of this project is to determine at the genomic level how events of mutation and reversion affect the phenotypes of a heat stable strain of mengovirus. Mutants representative of five phenotypes, heat stability, hemagglutination, polymerase production, pathogenicity and temperature-sensitivity will be selected from the mutant collection, and revertants will be isolated. The differences in nucleotide sequence between the mutants, revertants and the wild-type strain will be determined using recombinant DNA techniques. Hybrid infectious cDNA or RNA transcripts will be utilized to specifically identify and analyze the ts mutations and reversions. Site-directed mutagenesis will be used to confirm and extend the results obtained with the hybrid infectious cDNAs or RNA transcripts. Interpretation of the data will lead to a better understanding of the regulation of expression of these viral-specific functions. This project has significance in understanding genetic regulation of physiological properties of positive-stranded viruses. Further, the project has relevance in the development of safe, effective vaccines, and in the transmission of virus from tissue to tissue during a viremia.